Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils

Abstract

Plants require nitrogen (N) to make proteins, nucleic acids and other biological molecules. It is widely accepted that plants absorb inorganic forms of N to fill their needs. However, recently it has become clear that plants also have the capacity to absorb organic N from soils. In this paper we describe a new kind of symbiosis involving seed-vectored rhizobacteria and grasses that is targeted at enhancing acquisition of organic N from soils. Our proposal is based on results of experiments on seedlings of grass species Festuca arundinacea Schreb., Lolium perenne L. and Poa annua L. that suggest: (i) seed-vectored rhizobacteria colonize seedling roots and influence their development; (ii) reactive oxygen secretion by seedling roots plays a role in organic N procurement by denaturing microbial proteins in the vicinity of roots (daytime activity); and (iii) plant root and microbial proteases degrade denatured proteins prior to absorption by roots (night-time activity). This research involved the following types of studies: (i) seedling root development experiments with and without rhizobacteria on a variety of substrates in agarose media and (ii) isotopic N-tracking experiments to evaluate the absorption into seedlings of N obtained from degradation of proteins. We hypothesize that grasses, in particular, are adapted to scavenge organic N from soils through application of this 'oxidative nitrogen scavenging' symbiosis with rhizobacteria, and their soil-permeating root systems. This newly discovered symbiosis in grass species could lead to new ways to cultivate and manage grasses to enhance efficiency of N utilization and reduce applications of inorganic fertilizers.

Keywords: Grasses; microbiome; nitrogen use efficiency; oxidative nitrogen scavenging; plant growth-promoting rhizobacteria; symbiosis.

Figure 1.

Bacteria on root hairs of cool-season grass seedlings; stained with DAB/peroxidase for reactive oxygen (brown) and counterstained with aniline blue/lacto-phenol for protein. (A) Bacteria (arrow) on surface of root hair of L. perenne seedling. (B) Bacteria and bacterial protein (arrows) on surface of root hair of P. annua seedling. (C) Bacteria (arrows) on surface of root hair of P. annua seedling. (D) Bacteria (small arrows) and denatured proteins (large arrows) on the surface of root hair of P. annua seedling.

Figure 2.

Poa annua seedlings showing reactive oxygen (H₂O₂) staining around roots. (A) Seedlings growing on 0.7 % agarose showing diffuse zones of reactive oxygen (brown) around roots. (B) Seedlings growing on 0.1 % albumin agarose showing dense zones of reactive oxygen (arrows) around roots. (C) Root surface showing root hairs and layer of bacteria (arrow). (D) Root without bacteria growing in 0.7 % water agarose medium, showing absence of root hairs. (E) Root with bacteria growing on 0.7 % water agarose medium, showing reactive oxygen zone and root hairs (arrows).

Figure 3.

Poa annua root hairs stained for reactive oxygen. (A) Root hair showing denatured cellulase enzyme aggregation (arrow) on root hair with high reactive oxygen staining on protein as evidenced by darker brown colour. (B) Root hair showing the smoothing effect of the cellulase particle (arrow) in the area of close contact with the root hair. (C) Root hair without protein or bacteria. (D) Root hair showing adherent albumin protein (arrows).

Figure 4.

Results of ¹⁵N-labelled-protein absorption experiment showing enhanced ¹⁵N absorption due to rhizobacteria and reactive oxygen effects. All values expressed as mean ± standard error of mean; means with the same letter are not significantly different according to the Duncan's multiple range test (P < 0.05). The y-axis is the level of protein absorption (expressed as δ¹⁵N vs. air) and the x-axis shows the six treatments. The numbers above bars are means of the δ¹⁵N vs. air values and the bars show standard errors. The highest ¹⁵N incorporation (5415.46) was seen in shoots of seedlings bearing bacteria grown on agarose containing ¹⁵N-labelled protein in a 12-h alternating light/dark cycle (treatment = protein, bacteria, light/dark). Less ¹⁵N incorporation (3817.49) was seen in shoots of seedlings grown under the same conditions but without bacteria (treatment = protein, no bacteria, light/dark). Slightly less ¹⁵N incorporation (2488.49) was seen in shoots of seedlings grown on labelled proteins but without bacteria in total darkness to suppress reactive oxygen secretion (protein, no bacteria, dark). Minimal ¹⁵N incorporation was seen into shoots of seedlings grown on agarose that did not contain ¹⁵N-labelled protein (treatments = no protein, bacteria, light/dark; no protein, no bacteria, light/dark; no protein, no bacteria, dark).

Figure 5.

Proposed cyclic model of the oxidative nitrogen scavenging process in grasses. In daylight grasses secrete hydrogen peroxide from roots in order to denature proteins (microbial exoenzymes) around roots. At night plant and microbial proteases degrade oxidized proteins to form smaller peptides or oligopeptides that may be absorbed by roots.